Mutagenized IL 13-based chimeric molecules

ABSTRACT

This invention provides mutagenized interleukin 13 molecules that show improved specificity for the restricted (IL4 independent) IL13 receptor and reduced cross-reactivity with the IL4/IL4 shared receptor. The mutagenized IL13 molecules include one or more mutations in a domain that interacts with the 140 kDa hIL4Rβ or the hIL13Rα 1  subunit. These mutagenized IL13 molecules provide effective targeting moieties in chimeric molecules (e.g. fusion proteins) that specifically deliver effector molecules (e.g. cytotoxins) to cells overexpressing IL13 receptors (e.g. cancer cells such as gliomas).

BACKGROUND OF THE INVENTION

In a chimeric molecule, two or more molecules that exist separately intheir native state are joined together to form a single entity(molecule) having the desired functionality of all of its constituentmolecules. Frequently, one of the constituent molecules of a chimericmolecule is a “targeting molecule”. The targeting molecule is a moleculesuch as a ligand or an antibody that specifically binds to itscorresponding target, for example a receptor on a cell surface. Thus,for example, where the targeting molecule is an antibody, the chimericmolecule will specifically bind (target) cells and tissues bearing theepitope to which the antibody is directed.

Another constituent of the chimeric molecule may be an “effectormolecule.” The effector molecule refers to a molecule that is to bespecifically transported to the target to which the chimeric molecule isspecifically directed. The effector molecule typically has acharacteristic activity that is desired to be delivered to the targetcell. Effector molecules include cytotoxins, labels, radionuclides,other ligands, antibodies, drugs, prodrugs, liposomes, and the like.

In particular, where the effector component is a cytotoxin, the chimericmolecule may act as a potent cell-killing agent specifically targetingthe cytotoxin to cells bearing a particular target molecule. Forexample, chimeric fusion proteins which include interleukin 4 (IL4) ortransforming growth factor (TGFα) fused to Pseudomonas exotoxin (PE),interleukin 2 (IL2) fused to Diphtheria toxin (DT) have been shown tospecifically target and kill cancer cells (Pastan et al., Ann. Rev.Biochem., 61: 331-354 (1992)).

The targeting moiety of these chimeric cytotoxins is often selected topacifically target and bind to growth factor receptors, particularlythose receptors that are overexpressed on cancer cells as compared tonormal cells (e.g., Debinski et al. (1993) J. Biol. Chem., 268:14065-14070, Phillips et al. (1994) Cancer Res., 54: 1008-1015, Debinskiet al. (1994) Int. J. Cancer, 58: 744-748). However, even where thetarget receptor is overexpressed on cancer cells there is typically asignificant level of receptor expression on normal cells as well.Therefore, even though one can obtain a therapeutic window for thecytotoxins, toxicities related to the presence of growth factorreceptors on normal cells are dose-limiting for their administration(Phillips et al. (1994) Cancer Res., 54: 1008-1015, Debinski et al.(1994) Int. J. Cancer, 58: 744-748). It is thus desirable to identifytargets or targeting ligands that show provide increased specificity forcancer cells as compared to normal cells and thereby improve the dosagelevels that can be administered with diminished or no toxicside-effects.

SUMMARY OF THE INVENTION

This invention provides novel targeting ligands (specific bindingmoieties) that have increased specificity for cancer cells as comparedto normal cells and therefore extremely effective for specificallydelivering effector molecules to various neoplasias. The targetingligands are mutagenized IL13 molecules having one or more mutations inthe domain that interacts with the hIL4 receptor subunit designated the140 kDa hIL4R_(β) subunit.

Particularly preferred mutagenized IL13 molecules (specific bindingmoieties) of this invention are mutagenized human IL13 molecules. Thesemolecules can be mutated at one or more of a variety of residuesincluding at residues 12, 13, 14, 65, 66, 67, 68, 69, 70, 109, or 112.In one particular embodiment, residue 13 is a basic amino acid. Otherpreferred mutagenized IL13 molecules include lysine or arginine atresidue 13 and/or aspartic acid at residue 66 and/or aspartic acid atresidue 69 and or aspartic acid at residue 109 or 112. When a nativehuman IL13 is mutated these mutations can include hIL13.E13R,hIL13.R66D, hIL13.S69D, hIL13.E13K, hIL13.R109D, and hIL13.R112D.Preferred double mutations include hIL13.E13K/R66D or hIL13.E13K/S69D.The specific binding moieties (mutagenized IL13) can be attached to andtherefore comprise an effector molecule as described herein.

In one embodiment, any of the mutagenized IL13 molecules describedherein is a component of a chimeric molecule having the formula:

R¹—(L)_(j)—(R²)_(n)

in which R¹ is the mutagenized human interleukin 13, j and n areindependently 0 or 1; R² is an effector molecule; and L is an optionallinker. The effector molecule can be virtually any molecule that can beattached to the mutagenized IL 13. Effector molecules include, but arenot limited to cytotoxins, labels, antibodies, liposomes, lipids, DNA orRNA nucleic acids, DNA or RNA vector, recombinant viruses,chemotherapeutics, anti-cancer antibiotics, photosensitizers, and thelike. Particularly preferred cytotoxins include a Pseudomonas exotoxinor a Diphtheria toxin. The Pseudomonas exotoxin can be modified suchthat it substantially lacks domain Ia, and most preferred Pseudomonasexotoxins include PE38QQR and PE4E. It will be appreciated that theeffector molecule can be attached to either the amino terminus, thecarboxyl terminus or to an internal residue of the mutagenized IL13molecule although terminal attachment is preferred.

Preferred cytotoxic chimeric molecules are fuision proteins and includeany of the mutagenized IL13 molecules described herein fused to thecytotoxin. Particularly preferred cytotoxins include any of the abovemutagenized IL13 molecules fused to a Pseudomonas exotoxin (e.g.,hIL13.E13K-PE38QQR, hIL13.E13K-PE4E, etc.). Particularly preferredcytotoxic molecules include, but are not limited to hIL13.E13K-PE38QQR,hIL13.E13K-PE4E, hIL13.R66D-PE38QQR, hIL13.R66D-PE4E,hIL13.S69D-PE38QQR, hIL13.S69D-PE4E, hIL13.R109D-PE38QQR,hIL13.R112D-PE38QQR, hIL13.R109D-PE4E, hIL13.R112D-PE4EhIL13.E13K/R109D-PE38QQR, hIL13.E13K/R112D-PE38QQR,hIL13.E13K/R66D-PE4E, hIL13.E13K/RS69D-PE4E, DT390-hIL13.E13K,DT390-hIL13.R66D, DT390-hIL13.S69D, DT390-hIL13.R109D,DT390-hIL13.R112D, DT390-hIL13.E13K/R109D, and DT390-hIL13.E13K/R112D.

In another embodiment, this invention provides methods of delivering aneffector molecule to a cell bearing an interleukin 13 receptor (IL13R).The methods involve contacting the cell with a chimeric moleculecomprising the effector molecule attached to any of the mutagenizedinterleukin 13 (IL13) molecules described herein. The methods caninvolve any of the chimeric molecules described herein.

Where the effector molecule is a cytotoxin, this invention providesmethods of killing a cell or inhibiting the growth (and/orproliferation) of a cell expressing an IL13 receptor (IL13R). Again,these methods involve contacting the cell any of the mutagenizedIL13-cytotoxin chimeric molecules described herein. In a preferredembodiment, the cell is a neoplastic cell (e.g. a glioma).

The cytotoxic chimeric molecules described herein can be used ascomponents of a pharmacological composition. In this embodiment, thecomposition comprises any one or more of the cytotoxic chimericmolecules of this invention and a pharmacologically acceptableexcipient,

The mutagenized IL13 can also be attached to a detectable label. Thechimeric label can be used to detect and/or localize and/or quantify acell or cells expressing an IL13 receptor. The label when administeredto a subject will localize at the site(s) of cells expressing oroverexpressing IL13 receptors and detection of the label provides anindication of the presence, absence, quantity or location of such cells.Similarly ex vivo detection can be accomplished e.g. using a biologicalsample taken from the subject.

This invention also provides kits for the detection of cells expressingIL13 receptors or for inhibiting the growth and/or proliferation of suchcells. The kits preferably include one or more containers containing amutagenized IL13 of this invention. The mutagenized IL13 can be attachedto either label (e.g. for detection of an IL13R bearing cell) or acytotoxin (e.g. for inhibiting the growth of an IL13R bearing cell). Anyof the cytotoxic or label (or other) chimeric molecules of thisinvention can be included in the kit.

DEFINITIONS

The term “specifically binds”, as used herein, when referring to aprotein or polypeptide, or receptor refers to a binding reaction whichis determinative of the presence of the protein or polypeptide orreceptor in a heterogeneous population of proteins and other biologics.Thus, under designated conditions (e.g. immunoassay conditions in thecase of an antibody), the specified ligand or antibody binds to itsparticular “target” (e.g an IL13 specifically binds to an IL13 receptor)and does not bind in a significant amount to other proteins present inthe sample or to other proteins to which the ligand or antibody may comein contact in an organism.

The hIL4 receptor subunit designated the 140 kDA hIL4R_(β) subunitrefers to a polypeptide that is common to a shared IL13/IL4 receptor andall other IL4 receptors on “normal” (non-neoplastic cells) such as HUVEC(endothelial cells) (see, e.g., Idzerda et al. (1990) J. Exp. Med., 171:861-873).

The phrase “a domain that interacts (or specifically interacts) with thehIL13/IL4 receptor subunit designated the 140 kDA hIL4R_(β) subunit”, asused herein, refers to a domain of a polypeptide (e.g. IL13) disruptionof which reduces or eliminates binding of an IL13 to the IL13/IL4receptor or that reduces or eliminates effector activity (e.g. cytotoxicactivity) of a chimeric molecule having the disrupted IL13 molecule on acell or cells that express the 140 kDa hIL4Rβ, subunit (e.g., HUVECendothelial cells). Alteration of one or more amino acids in the domainpreferably diminishes or eliminates interaction with cells expressingthe 140 kDA hIL4R_(β) subunit but shows improvement in the interactionof the IL13 or IL13 chimeric molecule on cells over-expressingrestrictive IL4R-independent IL13 binding sites (e.g., on gliomas).

A mutation in a polypeptide refers to the substitution of an amino acidat a particular position in a polypeptide with a different amino acid atthat position. Thus, for example, the mutation hIL13.E13K indicates thatthe native amino acid at position 13 in IL13 (glutamic acid, E) isreplaced with lysine (K). The “mutation” does not require an actualremoval and substitution of the amino acid(s) in question. The proteincan be created de novo with the “replacement” amino acid in theposition(s) of the desired mutation(s) so the net result is equivalentto the replacement of the amino acid in question.

A “mutagenized IL13 ” or “mutagenized hIL13” refers to an IL13 in whichone or more of the amino acids differ from the corresponding amino acidsin the native form of the IL13. Thus, for example, where a native humanIL13 has a glutamic acid at position 13, a mutagenized human IL13 canhave an amino acid other than glutamic acid at position 13 (e.g.,glutamic acid is substituted with lysine). It will appreciated thatmutagenized IL13 molecules of this invention include mutagenized IL13molecules of other mammalian species (e.g., rat, murine, porcine,largomorph, non-human primates, bovine, canus, and the like) and thisinvention contemplates the use of chimeric molecules in veterinary aswell as human medical conditions.

A chimeric molecule, as used herein refers to a molecule in which, twoor more molecules that exist separately in their native state are joinedtogether to form a single entity (molecule) having the desiredfunctionality of all of its constituent molecules. Preferred chimericmolecules of this invention involve one or more IL13 (more preferably amutagenized human IL13) joined to one or more effector molecules. Themutagenized IL13 acts as a targeting molecule preferably binding thechimeric molecule to cells expressing or overexpressing a restrictiveIL4R-independent IL13 receptor (IL13R).

A fusion protein as used herein is a chimeric molecule in which thecomponents making up the chimeric molecule are polypeptides and thepolypeptides are joined directly (or through a peptide linkage) viapeptide bonds. The fusion protein thus forms a continuous singlepolypeptide having domains corresponding to the different (e.g.,targeting and effector) components.

A “specific binding moiety” or a “targeting moiety” refers to a molecule(e.g., a polypeptide) that specifically binds to a particular target.Thus, for example, an interleukin-13 (IL13) is a specific binding moietythat specifically binds to an IL13 receptor (although it will berecognized that where the IL13 receptor shares a component with an IL4receptor) the specific binding moiety may cross-react with the IL4receptor. Nevertheless the binding moiety is still regarded as specificbecause its interaction is specific to these two components and it doesnot generally bind to any protein found in the organism or biologicalsample. Preferred specific binding moieties of this inventionpreferentially bind to the restrictive IL4R-independent tumor associatedIL13 receptor rather than the IL4 receptor and typically show an avidityand/or specificity for an IL13 receptor at least 1.5-fold, preferably atleast 2-fold, more preferably at least 5-fold, and most preferably atleast 10-fold or even at least 100-fold greater than its affinity and/orspecificity for an IL4 receptor.

An “effector molecule” as used herein, refers to a molecule that it isdesired to deliver to a particular target (e.g., to a target cell). Theeffector molecule preferably has a characteristic activity that isdesired to be delivered to the target cell. Effector molecules includecytotoxins, labels, radionuclides, other ligands, antibodies, drugs,prodrugs, liposomes, lipids, recombinant viruses, chemotherapeutics,anti-cancer antibiotics, photosensitizers, and the like. It will beappreciated that some effectors once delivered to the cell arepreferentially internalized while others (such as labels) need not beinternalized. However many effectors (e.g., PE, or DT cytotoxins) aremore effective on internalization.

The term “delivering an effector molecule to a cell” refers topreferentially binding such that when an organism is systemicallytreated with a chimeric molecule comprising a mutagenized IL13 of thisinvention, or when a cell culture is treated with a chimeric moleculecomprising a mutagenized IL13 of this invention, the chimeric moleculepreferentially accumulates adjacent to or on the target cell or ispreferentially internalized by the cell as compared to cells lacking orhaving a lower concentration of the target to which the mutagenized IL13is directed (e.g. the IL13 receptor).

The term “inhibiting the growth of a cell” refers to inhibition ofgrowth and/or proliferation of a cell or cells. Such inhibition mayinvolve killing of one or more cells. Methods of assaying cell growthand/or proliferation are well known to those of skill in the art.

The terms “isolated” or “substantially purified” or “isolated” whenreferring to a protein, means a chemical composition which isessentially free of other cellular components. It is preferably in ahomogeneous state although it can be in either a dry or aqueoussolution. Purity and homogeneity are typically determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A proteinwhich is the predominant species present in a preparation issubstantially purified. Generally, a substantially purified or isolatedprotein will comprise more than 80% of all macromolecular speciespresent in the preparation. Preferably, the protein is purified torepresent greater than 90% of all macromolecular species present. Morepreferably the protein is purified to greater than 95%, and mostpreferably the protein is purified to essential homogeneity, whereinother macromolecular species are not detected by conventionaltechniques. The term “purifying” when used in reference to a protein ora receptor refers to rendering the protein or receptor in such anisolated or substantially purified state.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers.

The term “nucleic acid” refers to a deoxyribonucleotide orribonucleotide polymer in either single- or double-stranded form, andunless otherwise limited, encompasses known analogs of naturalnucleotides that can function in a similar manner as naturally occurringnucleotides. The term also include nucleotides linked by peptidelinkages as in “peptide” nucleic acids.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic model of hIL13/hIL4R and the effect ofmutagenized interleukin 13 on it.

FIG. 2 shows a schematic drawing of interleukin 13 and interleukin 4.

FIG. 3 shows the N-termini of both human and murine interleukin 13 andinterleukin 4 ending at the first α-helix, α-helix A. Amino acids whichform the α-helix A are underlined. The conserved residue of glutamicacid is typed in boldface font.

FIG. 4 illustrates the proliferative activities of hIL13, hIL13.E13K,and hIL4.Y124D on TF-1 cells. hIL4.Y124D was not used at a concentrationof 0.01 ng/ml in these assays. The vertical bars represent SDs and maybe smaller than the symbols.

FIG. 5 illustrates the cytotoxic activity of hIL13-PE4E andhIL13.E13K-PE4E on HUVEC. The vertical bars represent SDs and may besmaller than the symbols.

FIGS. 6A, 6B, and 6C illustrate the cytotoxic effect of hIL13.E13K-PE4Eor hIL13-PE4E on SNB-19 cells (FIG. 6A) and U-251 MG cells (FIG. 6B).Neutralization of the cytotoxicity of hIL13.E13K-PE4E by hIL13,hIL13.E13K, and hIL4 on SNB-19 cells (FIG. 6C). hIL4 was not added tocells that were treated with the lowest concentration of the cytotoxin.The vertical bars represent SDs (A and B) and may be smaller than thesymbols.

FIG. 7 illustrates competition for the binding sites for ¹²⁵I-hIL13 byhIL13.E13K and hIL13 on U-251 MG cells.

FIGS. 8A and 8B show the anti-tumor activities of hIL13.E13K and hIL13cytotoxins on a human U251-MG glioma tumor growing in mice. Thearrowheads indicate the days of cytotoxins injections. The vertical barscorrespond to the SEs and may be smaller than the symbols (not shown forhIL13-PE4E 2.0 μg in 8A).

DETAILED DESCRIPTION

This invention provides ligands that are highly specific to the IL13receptor and, when incorporated into chimeric molecules (e.g., chimericfusion proteins) are capable of specifically directing the chimericmolecules to cells expressing IL13 receptors. Since IL13 receptortargets are characteristically overexpressed on cancer cells, thetargeting agents of this invention are particularly useful forspecifically directing agents to those cancer cells (e.g., gliomas). Ina preferred embodiment, the ligands are mutagenized IL13 molecules, inparticular, IL13 molecules containing one or more mutations in a domainthat interacts with the hIL13/hIL4 receptor subunit designated the 140kDA hIL4R_(β) subunit.

The target IL13 receptors are growth factor receptors that show a numberof similarities to the IL4 receptors. Studies of the similarities anddifferences between the IL13 receptor (IL13R) and the IL4 receptor(IL4R) suggest that IL13 binds to the IL4 receptor (as well as to theIL13 receptor) and that IL13 binding to the IL4 receptor is fullycompeted for by IL4 (Zurawski et al. (1995) J. Biol. Chem., 270:13869-13878, Vita et al. (1995) J. Biol. Chem., 270: 3512-3517, and Tonyet al. (1994) Eur. J. Biochem., 225: 659-665)).

A recently proposed model for the hunan IL13 receptor suggests that itis heterodimeric and comprises an hIL13 binding protein (Debinski et al.(1995) J. Biol. Chem., 270: 16775-16780), Obiri et al. (1995) J. Biol.Chem. 270: 8797-8804, Caput et al. (1996) J Biol. Chem. 271:16921-16926; Hilton et al. (1996) Proc. Natl. Acad. Sci., USA, 93:497-501) and a 140 kDa hIL4R_(β) (Obiri et al. (1996) Clin. Cancer Res.2: 1743-1749) (FIG. 1). The latter is a subunit shared with the hIL4receptor (Debinski et al. (1996) J. Biol. Chem., 271: 22428-22433,Zurawski et al. (1995) J. Biol. Chem., 270: 13869-13878, Vita et al.(1995) J. Biol. Chem., 270: 3512-3517, and Tony et al. (1994) Eur. J.Biochem., 225: 659-665, Hilton et al. (1996) Proc. Natl. Acad. Sci. USA,93: 497-50 1). Thus, human interleukin-13 (hIL13) may contain at leasttwo receptor interaction sites (domains): (i) one which recognizes the140 kDa hIL4R_(β) subunit, and (ii) another site which interacts withits proper binding proteins (Obiri et al. (1995) J. Biol. Chem. 270:8797-8804, Caput et al. (1996) J Biol. Chem. 271: 16921-16926; Hilton etal. (1996) Proc. Natl. Acad. Sci., USA, 93: 497-501). These putativesites are proposed here based on structural homology to hIL4 and thebelief that hIL13 exists as a compact core-bundle of the fouranti-parallel a-helices cytokine (FIG. 2).

A predictive model of hIL13 is described by Bamborough et al. (1994)Prot. Eng., 7: 1077-1082. This model is analogous to the model of hIL4which proposes that IL4 binds the 140 kDa hIL4R_(β) through one site andthe γ_(c) , subunit through another site which produces heterodimerichigh affinity hIL4R (Russell et al. (1993) Science, 262: 1880-1883). Amutation of glutamic acid at position 9 to lysine in hIL4 (hIL4.E9K)severely impairs binding of hIL4 to the 140 kDa hIL4R_(β) (Kruse et al.(1993) EMBO J. 12: 5121-5129) (FIG. 3).

Recently, it was demonstrated that human (h) gliomas express largenumber of receptors (R) for interleukin 13 (IL13) (Debinski et al.(1995) Clin. Cancer Res. 1: 12531-1258). It was also shown that both IL4and an antagonist of hIL4, hIL4.Y124D, which binds the 140 kDa hIL4Rβ-chain protein and block the effects of hIL13 and hIL4 on normal cells,did not block the binding and internalization of IL13 in glioma cellsunlike on normal cells and some adenocarcinomas (Debinski et al. (1995)Clin. Cancer Res. 1: 1253-1258; Debinski et al. (1996) J. Biol. Chem.271: 22428-224; Debinski et al. (1995) J. Biol. Chem. 270; 16775-16780). These and other findings demonstratc the existence of hIL13 receptors(e.g., on cancers) that do not interact with IL4 and presumably do notinvolve the 140 kDa hIL4R β-chain (hIL4Rβ)

This was demonstrated by the observation that the use of hIL4 andhIL4.Y124D in conjunction with IL13R directed chimeric moleculesenhanced the specificity of these molecules to cells bearing the IL13receptor.

It is demonstrated herein that a similar effect to that exhibited byhIL4 and hIL4.Y124D can be obtained by mutagenizing IL13 itself andusing the mutagenized IL13 as a targeting moiety in a chimeric molecule.In one embodiment, cytotoxins are described herein in which thetargeting moiety (IL13) is mutagenized by changing glutamic acid atposition 13 to lysine (producing hIL13.E13K) and the toxic effectormolecule is a Pseudomonas exotoxin A (PE) derivative (e.g., PE38QQR, orPE4E).

It is also taught herein that by altering a putative binding site ofIL13 which interacts with the 140 kDa IL4 receptor β-chain, one canalter interaction of the cytotoxins (or other IL13R-directed chimericmolecules) with the IL13R and IL4R common elements that arepredominantly expressed in normal tissues. Indeed, it is demonstratedherein that, for example, hIL13.E13K-PE4E is less active on normalcells, such as endothelial cells, which do express elements common toboth hIL4 and hIL13R. Unexpectedly, the action of hIL13.E13K-PE4E wasconsiderably more potent on human glioma cells when compared with thatcontaining the wild-type hIL13. Toxicities of the hIL13.E13K-basedcytotoxins in vivo are also several times lower when compared withchimeric cytotoxins utilizing unmutagenized hIL13 as a targeting moiety.Thus, it is demonstrated herein that a mutation in the domain of IL13that interacts with the hIL4 receptor subunit designated the 140 kDahIL4R_(β) subunit (e.g., a mutation at IL13 residue 13) makes a chimericcytotoxin less active on normal cells and, surprisingly, much moreactive on glioma cells. The increase in an overall specific cytotoxicactivity can be as high as 100-fold. Thus, hIL13 is amenable toengineering which leads to a much more discriminate recognition of thehIL13R that is expressed on cancer cells from the one present on normalcells.

As explained below, in a preferred embodiment the mutagenized IL13 canbe provided as a component of a chimeric molecule. Alternatively, themutagenized IL13 may be provided alone to bind to and therebyspecifically block the IL13 receptor.

I. Uses of Chimeric Molecules Targeted to the IL13 Receptor

Using the mutagenized IL13 molecules, this invention provides in oneembodiment, methods for specifically delivering an effector molecule toa cell bearing an IL13 receptor (e.g., a tumor cell such as a glioma).These methods utilize chimeric molecules comprising a mutagenized IL13(targeting molecule) attached to an effector molecule. The chimericmolecules of this invention specifically target tumor cells (especiallyglioma cells) while providing reduced binding to non-target cells ascompared to other targeted chimeric molecules known in the art.

This allows specific delivery of any of a number of effector moleculesto the target cell(s). Where the effector molecule is a cytotoxin, thisinvention provides for methods and compositions for impairing the growthand/or proliferation of cells (e.g., free cells or cells in tumors). Thechimeric cytotoxin is administered to an organism containing IL13receptor-bearing cells (e.g., cancer cells) which are then contacted bythe chimeric molecule. The mutagenized IL13 component of the chimericmolecule specifically binds to the overexpressed IL13 receptors on thecells. Once bound to the IL13 receptor on the cell surface, thecytotoxic effector molecule mediates internalization into the cell wherethe cytotoxin inhibits cellular growth or kills the cell. The cytotoxinmay be a native or modified cytotoxin such as Pseudomonas exotoxin (PE),Diphtheria toxin (DT), ricin, abrin, and the like.

In another embodiment, the chimeric molecules of this invention canprovide compositions and methods for detecting the presence or absenceof tumor cells. These methods involve providing a chimeric moleculecomprising an effector molecule, that is a detectable label attached toa mutagenized IL13. The mutagenized IL13 specifically binds the chimericmolecule to IL13R bearing target cells (e.g., tumor cells) which arethen marked by their association with the detectable label. Subsequentdetection of the cell-associated label indicates the presence of thetarget cell.

In yet another embodiment, the effector molecule may be another specificbinding moiety such as an antibody, a growth factor, or a ligand. Thechimeric molecule will then act as a highly specific bifunctionallinker. This linker may act to bind and enhance the interaction betweencells or cellular components to which the fusion protein binds. Thus,for example, where the chimeric molecule comprises a mutagenized IL13 ofthis invention attached to an antibody or antibody fragment (e.g. an Fvfragment of an antibody), the mutagenized IL13 specifically binds targetcells (e.g., cancer cells), while the effector component binds receptors(e.g., IL-2 or IL-4 receptors) on the surface of immune cells. Thechimeric molecule may thus act to enhance and direct an immune responsetoward target cancer cells. Alternatively, the mutagenized IL13 can beattached to a bacterial superantigen such as Staphylococcal EnterotoxinA and B (SEA and SEB), or other superantigens and can thereby activateimmune cells which will target a response to the cells (e.g., gliomacells) bearing the chimeric molecule.

In still yet another embodiment the effector molecule may be apharmacological agent (e.g. a drug) or a vehicle containing apharmacological agent. Thus the mutagenized IL13s of this invention maybe conjugated to a drug such as vinblastine, doxorubicin, genistein (atyrosine kinase inhibitor), an antisense molecule, ribozymes, and otherpharmacological agents known to those of skill in the art, therebyspecifically targeting the pharmacological agent to target cells overexpressing IL13 receptors.

Alternatively, the mutagenized IL13 may be bound to a vehicle containingthe therapeutic composition. Such vehicles include, but are not limitedto liposomes, lipids, micelles, various synthetic beads, and the like.

One of skill in the art will appreciate that the chimeric molecules ofthe present invention may include mutagenized IL13 molecules bound to asingle effector or conversely, multiple effector molecules bound to asingle mutagenized IL13 molecule. Thus, one embodiment one effector maybe bound to the mutagenized IL13 amino terminus while another effectoris bound to the mutagenized IL13 carboxyl terminus. The two effectorscan be the same or different.

In still other embodiment, the chimeric molecules may include bothmultiple mutagenized IL13 molecules and multiple effector molecules.Thus, for example, this invention provides for “dual targeted” cytotoxicchimeric molecules in which the mutagenized IL13 is attached to acytotoxic molecule and another molecule (e.g. an antibody, or anotherligand) is attached to the other terminus of the toxin. Such adual-targeted cytotoxin might comprise a mutagenized IL13 substitutedfor domain Ia at the amino terminus of a PE and anti-TAC(Fv) inserted indomain III, between amino acid 604 and 609. Other antibodies may also besuitable.

II. Mutagenized Interleukin 13 (IL13)

It was a discovery of this invention that mutagenized IL13 provides aligand having improved specificity for cells expressing a restrictive(IL4 receptor independent) IL13 receptor. Moreover, because themutagenized IL13 does not significantly bind to the 140 kDa hIL4R_(β)subunit that is shared by both the IL13 receptor (on non-neoplasticcells) and the IL4 receptor, the mutagenized IL13 shows reduced bindingto normal cells expressing the IL13/IL4 receptor. Particularly preferredmutagenized IL13 ligands of this invention have one or more mutations inthe domain that interacts with 140 kDa hIL4R_(β) subunit as describedbelow.

Native interleukin-13 (IL13) is a pleiotropic cytokine that isrecognized to share many of the properties of IL4. IL13 hasapproximately 30% sequence identity with IL-4 and exhibits IL4-likeactivities on monocytes/macrophages and human B cells (Minty et al.,Nature, 362: 248 (1993), McKenzie et al. Proc. Natl. Acad. Sci. USA, 90:3735 (1987)).

The nucleic acid and amino acid sequences of IL13 are well characterized(see, e.g. SWISS-PROT: P35225, McKenzie et al. (1993) Proc. Natl. Acad.Sci. USA, 90: 3735-3739) and either the polypeptide or nucleic acidsequence information can be used for the production of mutagenized IL13as described below and in Example 1.

A) Preferred Mutagenized IL13 molecules.

In a preferred embodiment, the mutagenized IL13 molecules of thisinvention show diminished interaction with the shared IL13/IL4 receptorand the same or improved interaction (e.g. binding or receptor mediatedactivity) with the restrictive (IL4R independent) IL13 receptor. Asexplained above, this is accomplished by mutagenizing the IL13 to reduceor eliminate interaction of the mutagenized IL13 (or chimeric molecule)with the 140 kDa IL4R_(β) subunit. This is preferably accomplished byintroducing mutations in the IL13 domain that interacts with the 140 kDaIL4R_(β).

Preferred mutations are thus located at in α-helix A and C. Preferredmutagenized IL13 molecules include mutations of one or more of residue13 and/or residue 66 and/or residue 69 and/or residue or residues 12,14, 65, 67, 68, 70, and 76. Particularly preferred mutations include oneor more of the following mutations: mutation of residue 13 to lysine orarginine, mutation of residue 66 to aspartic acid, mutation of residue69 to aspartic acid or mutation of residue 109 or 112 to aspartic acid.

Another mutagenizing strategy is to identify hIL13 mutants that aredeprived of interaction with their proper binding protein (e.g.,hIL13Rα¹) which is the other subunit of the shared IL13/IL4 receptor(FIG. 1 and Miloux et al. (1997) FEBS Letts., 401: 163-166). This isconsistent with IL13 having two receptor recognition sites. Suchmutations can include hIL13.R109D, hIL13.R112D, hIL13.F113D, etc. Othersuitable mutagenized IL13 molecules can be routinely identified usingthe methods described below.

B) Screening for Mutagenized IL13 Molecules.

As indicated above, preferred mutagenized IL13 molecules have mutationsthat diminish or eliminate interaction with the human IL4 receptorsubunit designated the 140 kDa hILR_(β) subunit, while not diminishing,and even increasing, their specificity and avidity for a restrictive,cancer-asssociated IL13 receptor, especially for an IL13 receptor thatdoes not include the 140 kDa hILR_(β). Identifying such mutagenized IL13molecules generally involves first producing one or more mutagenizedIL13 molecules and then screening the mutagenized IL13 molecules toidentify those that do not interact with the shared IL4/IL13 receptor,but still bind to a restrictive (IL-4 receptor independent) IL13receptor.

i) Mutagenizing IL13

Mutagenized IL13 molecules for use in this invention can routinely beproduced and screened. Numerous means of mutagenizing polypeptides arewell known to those of skill in the art. Since the amino acid sequenceof native IL13 is fully known, mutagenized IL13 molecules can bechemically synthesized or recombinantly expressed.

Mutated IL13 polypeptides of this invention may be synthesized usingstandard chemical peptide synthesis techniques. Some IL13 muteins can besynthesized as a single polypeptide. Chemical synthesis may, however, befacilitated by separately synthesizing subsequences and then fusing thesubsequences by condensation of the amino terminus of one molecule withthe carboxyl terminus of the other molecule thereby forming a peptidebond. Techniques for solid phase synthesis of polypeptides are wellknown to those of skill in the art (see, e.g., Barany and Merrifield,Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis,Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, PartA., Merrifield, et al. J. Am. Chem. Soc., 85: 2149-2156 (1963), andStewart et al., Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co.,Rockford, Ill. (1984)).

Mutated IL13 can also be produced through recombinant expression of IL13encoding nucleic acids in which the nucleic acid is modified, randomlyor in a site-specific manner, to change (substitute), add to, or delete,some or all of the amino acids in the encoded polypeptide.Alanine-scanning mutagenesis is widely used to examine structurefunction relationships of polypeptides.

Site-specific mutations canbe introduced into the IL13-encoding nucleicacid by a variety of conventional techniques, well described in thescientific and patent literature. Illustrative examples include:site-directed mutagenesis by overlap extension polymerase chain reaction(OE-PCR), as in Urban (1997) Nucleic Acids Res. 25: 2227-2228; Ke (1997)Nucleic Acids Res., 25: 3371-3372, and Chattopadhyay (1997)Biotechniques 22:1054-1056, describing PCR-based site-directedmutagenesis “megaprimer” method; Bohnsack (1997) Mol. L Biotechnol. 7:181-188; Ailenberg (1997) Biotechniques 22: 624-626, describingsite-directed mutagenesis using a PCR-based staggered re-annealingmethod without restriction enzymes; Nicolas (1997) Biotechniques 22:430-434, site-directed mutagenesis using long primer-unique siteelimination and exonuclease III. Unique-site elimination mutagenesis canalso be used (see, e.g., Dang et al. (1992) Anal. Biochem., 200: 81).The production of muteins of biologically active proteins such as IFN-beta and IL-2 is described in detail in U.S. Pat. No. 4,853,332 and themutation of IL13 is described in Example 1.

Modified IL13 of this invention can be further produced by chemicalmodification methods, see, e.g., Belousov (1997) Nucleic Acids Res.25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19: 373-380;Blommers (1994) Biochemistry 33: 7886-7896, and the like.

ii) Screening the IL13 Muteins

The mutagenized IL13 molecules can then be screened to identify thosethat preferentially bind to the restrictive (IL4 receptor independent)IL13 receptor and show reduced or no interaction (e.g. binding with theIL13/IL4 receptor in general or specifically either the 140 kDa hIL4_(β)subunit or the 70 kDA IL13Rα¹). It is possible to assay for simplebinding (e.g., detecting the competition of mutagenized IL13 withbinding of labeled wild-type IL13 to an IL13 or IL4 receptor), or,alternatively, to assay for some biological activity (e.g., the activityof an effector molecule) mediated through the binding of the mutagenizedIL13 and/or the mutagenized IL13 chimeric molecule. A simple bindingassay is described in U.S. Pat. No. 5,614,191.

The data generally suggest that IL13 receptor on gliomas (e.g. celllines U-251 MG and SNB-19) is restricted in that it does not interactwith IL4. Thus these cells provide a good target for assaying forpositive IL13R binding or activity. Other cells such as HUVECendothelial cells express an IL13/IL4 receptor that appears to utilizethe common 140 kDa hIL4_(β) subunit and contain the 70 kDa IL13Rα¹subunit. These cells thus provide good targets for assaying reducedbinding or activity of the mutagenized IL13 molecules. Thus preferredmutagenized IL13 molecules or chimeric molecules of this invention showincreased activity on U-251 MG and SNB-19 glioma and similar cells andreduced binding or activity on HUVEC (endothelial) cells. This can beassayed in a proliferative assay (see, e.g., the proliferative assay onTF-1 cells that express the shared IL13/IL4 receptor described inExample 1).

In a particularly preferred embodiment, the mutagenized IL13 isexpressed in fusion with a cytotoxin (e.g. PE38QQR, PE4E, etc.) and thecytotoxic activity (e.g. inhibition of growth or proliferation or uptakeof labeled substrate such as amino acids, etc.) on the target cells isassayed. Methods of assaying such activity are well known to those ofskill in the art and are illustrated, in Example 1 and in U.S. Pat. No.5,614,191. Other suitable assays include the inhibition of RANTESinduction by TNF and IFNγ (Farfaing-Koka et al., (1995) J. I. 154:1870-1878).

It will be appreciated that such assays can be performed in single assayformat. Alternatively, such assays are amenable to “parallel” processingin a high throughput screening system e.g., using a BiaCore system toassay binding (see, e.g., Nang et al. (1997) Proc. Nat. Acad. Sci. USA,94:1654-1662).

High throughput assays for the presence, absence, or quantification ofprotein binding or cell activity are well known to those of skill in theart. Thus, for example, U.S. Pat. No. 5,559,410 discloses highthroughput screening methods for proteins, while U.S. Pat. Nos.5,576,220 and 5,541,061 disclose high throughput methods of screeningfor ligand/antibody binding.

In addition, high throughput screening systems are commerciallyavailable (see, e.g., Zymark Corp., Hopkinton, Mass.; Air TechnicalIndustries, Mentor, Ohio; Beckman Instruments, Inc. Fullerton, Calif.;Precision Systems, Inc., Natick, Mass, etc.). These systems typicallyautomate entire procedures including all sample and reagent pipetting,liquid dispensing, timed incubations, and final readings of themicroplate in detector(s) appropriate for the assay. These configurablesystems provide high throughput and rapid start up as well as a highdegree of flexibility and customization.

III. The Effector Molecule

As described above, the effector molecule component of the chimericmolecules of this invention may be any molecule whose activity it isdesired to deliver to cells that overexpress IL13 receptors.Particularly preferred effector molecules include cytotoxins such as PEor DT, radionuclides, ligands such as growth factors, antibodies,detectable labels such as fluorescent or radioactive labels, andtherapeutic compositions such as liposomes, lipids, and various drugs.

A) Cytotoxins.

Particularly preferred cytotoxins include Pseudomonas exotoxins,Diphtheria toxins, ricin, and abrin. Pseudomonas exotoxin and Diphtheriatoxin are most preferred.

i) Pseudomonas Exotoxin (PE).

Pseudomonas exotoxin A (PE) is an extremely active monomeric protein(molecular weight 66 kD), secreted by Pseudomonas aeruginosa, whichinhibits protein synthesis in eukaryotic cells through the inactivationof elongation factor 2 (EF-2) by catalyzing its ADP-ribosylation(catalyzing the transfer of the ADP ribosyl moiety of oxidized NAD ontoEF-2).

The toxin contains three structural domains that act in concert to causecytotoxicity. Domain Ia (amino acids 1-252) mediates cell binding.Domain II (amino acids 253-364) is responsible for translocation intothe cytosol and domain III (amino acids 400-613) mediates ADPribosylation of elongation factor 2, which inactivates the protein andcauses cell death. The function of domain Ib (amino acids 365-399)remains undefined, although a large part of it, amino acids 365-380, canbe deleted without loss of cytotoxicity (see Siegall et al. (1989) J.Biol. Chem. 264: 14256-142610.

Where the mutagenized IL13 is fused to PE, a preferred PE molecule isone in which domain Ia (amino acids 1 through 252) is deleted and aminoacids 365 to 380 have been deleted from domain lb. However all of domainIb and a portion of domain II (amino acids 350 to 394) can be deleted,particularly if the deleted sequences are replaced with a linkingpeptide.

In addition, the PE molecules can be further modified usingsite-directed mutagenesis or other techniques known in the art, to alterthe molecule for a particular desired application. Means to alter the PEmolecule in a manner that does not substantially affect the functionaladvantages provided by the PE molecules described here can also be usedand such resulting molecules are intended to be covered herein.

For maximum cytotoxic properties of a preferred PE molecule, severalmodifications to the molecule are recommended. An appropriate carboxylterminal sequence to the recombinant molecule is preferred totranslocate the molecule into the cytosol of target cells. Amino acidsequences which have been found to be effective include, sequences thatfunction to maintain or recycle proteins into the endoplasmic reticulum,referred to here as “endoplasmic retention sequences”. See, for example,Chaudhary et al, Proc. Natl. Acad. Sci. USA 87: 308-312 and Seetharam etal (1991) J. Biol. Chem. 266: 17376-17381.

Deletions of amino acids 365-380 of domain Ib can be made without lossof activity. Further, a substitution of methionine at amino acidposition 280 in place of glycine to allow the synthesis of the proteinto begin and of serine at amino acid position 287 in place of cysteineto prevent formation of improper disulfide bonds is beneficial.

In a preferred embodiment, the mutagenized IL13 targeting molecule isinserted in replacement for domain Ia Preparation of an analogousmolecule, IL13-PE38QQR, is described in U.S. Pat. No: 5,614,191. Inaddition, similar insertions have been accomplished in what is known asthe TGFα-PE40 molecule (also referred to as TP40) described in Heimbrooket al. (1990) Proc. Natl. Acad. Sci, USA, 87: 4697-4701 and in U.S. Pat.No. 5,458,878.

Preferred forms of PE contain amino acids 253-364 and 381-608, and arefollowed by the native sequence or mutant sequences described inChaudhary et al.(1990), Proc. Nat. Acad Sci. USA 87: 308-312. Lysines atpositions 590 and 606 may or may not be mutated to glutamine.

In a particularly preferred embodiment, the IL13 receptor targetedcytotoxins of this invention comprise the PE molecule designatedPE38QQR. This PE molecule is a truncated form of PE composed of aminoacids 253-364 and 381-608. The lysine residues at positions 509 and 606are replaced by glutamine and at 613 are replaced by arginine (Debinskiet al. (1994) Bioconj. Chem., 5: 40).

In another particularly preferred embodiment, the IL13 receptor targetedcytotoxins of this invention comprise the PE molecule designated PE4E.PE4E is a “full length” PE with a mutated and inactive native bindingdomain where amino acids 57, 246, 247, and 249 are all replaced byglutamates (see, e.g., Chaudhary et al. (1995) J. Biol. Chem., 265:16306).

The mutagenized IL13 targeting molecule may also be inserted at a pointwithin domain III of the PE molecule. In this instance, the mutagenizedIL13 molecule is preferably fused between about amino acid positions 607and 609 of the PE molecule. This means that the mutagenized IL13 isinserted after about amino acid 607 of the molecule and an appropriatecarboxyl end of PE is recreated by placing amino acids about 604-613 ofPE aaer the targeting molecule. Thus, the mutagenized IL13 is insertedwithin the recombinant PE molecule after about amino acid 607 and isfollowed by amino acids 604-613 of domain III. The mutagenized IL13 mayalso be inserted into domain Ib to replace sequences not necessary fortoxicity. Debinski, et al. Mol. Cell. Biol., 11: 1751-1753 (1991).

In a preferred embodiment, the PE molecules are fused to the targetingmolecule by recombinant means. The genes encoding protein chains may becloned in cDNA or in genomic form by any cloning procedure known tothose skilled in the art (see, e.g., Sambrook et al. (1989), MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory). Methods ofcloning genes encoding PE fused to various ligands are well known tolose of skill in the art (see, e.g., Siegall et al. (1989) FASEB J., 3:2647-2652; and Chaudhary et al. (1987) Proc. Natl. Acad. Sci. USA, 84:4538-4542).

Those skilled in the art will realize that additional modifications,deletions, insertions and the like may be made to the chimeric moleculesof the present invention or to the nucleic acid sequences encoding IL13receptor-directed chimeric molecules. Especially, deletions or changesmay be made in PE or in a linker connecting the mutagenized IL13 to PE,in order to increase cytotoxicity of the fusion protein toward targetcells or to decrease nonspecific cytotoxicity toward cells withoutantigen for the antibody. Such constructions may be made by methods ofgenetic engineering well known to those skilled in the art (see,generally, Sambrook et al., supra) and may produce proteins that havediffering properties of affinity, specificity, stability and toxicitythat make them particularly suitable for various clinical or biologicalapplications.

ii) Diphtheria Toxin (DT).

Like PE, diphtheria toxin (DT) kills cells by ADP-ribosylatingelongation factor 2 thereby inhibiting protein synthesis. Diphtheriatoxin, however, is divided into two chains, A and B, linked by adisulfide bridge. In contrast to PE, chain B of DT, which is on thecarboxyl end, is responsible for receptor binding and chain A, which ispresent on the amino end, contains the enzymatic activity (Uchida etal., Science, 175: 901-903 (1972); Uchida et al. J. Biol. Chem., 248:3838-3844 (1973)).

In a preferred embodiment, the targeting molecule-Diphtheria toxinfusion proteins of this invention have the native receptor-bindingdomain removed by truncation of the Diphtheria toxin B chain.Particularly preferred is DT388, a DT in which the carboxyl terminalsequence beginning at residue 389 is removed. Chaudhary, et al. (1991)Bioch. Biophys. Res. Comm., 180: 545-551.

Another preferred diphtheria toxin is DT390 a diphtheria toxin in whichthe native binding domain is eliminated and the L at position 390 isfollowed by SPGPVPPST of the mutagenized IL13.

Like the PE chimeric cytotoxins, the DT molecules may be chemicallyconjugated to the IL13 receptor targeting molecule, but, in a preferredembodiment, the targeting molecule will be fused to the Diphtheria toxinby recombinant means. The genes encoding protein chains may be cloned incDNA or in genomic form by any cloning procedure known to those skilledin the art. Methods of cloning genes encoding DT fused to variousligands are also well known to those of skill in the art (see, e.g.,Williams et al. J Biol. Chem. 265: 11885-11889 (1990)).

The term “Diphtheria toxin” (DT) as used herein refers to full lengthnative DT or to a DT that has been modified. Modifications typicallyinclude removal of the targeting domain in the B chain and, morespecifically, involve truncations of the carboxyl region of the B chain.A mutagenized binding domain may also be present.

iii) Other Toxins.

It will be appreciated that the chimeric molecules of this invention caninclude cytotoxins other than diphtheria toxin or Pseudomonas exotoxin.Many such cytotoxins are known to those of skill and include but are notlimited to ricin, abrin, saporin, pokeweed viral protein for virtuallyany other toxin that is capable of being conjugated or fused to apolypeptide.

B) Detectable Labels.

Detectable labels attached to the mutagenized IL13 molecules of thisinvention can be used in diagnostic assays (e.g., in the detection ofshed tumor cells overexpression the IL13 receptor) and/or in the in vivolocalization of tumor cells. Detectable labels suitable for use as theeffector molecule component of the chimeric molecules of this inventioninclude any composition detectable by spectroscopic, photochemical,biochemical, immunochemical, electrical, optical or chemical means.Useful labels in the present invention include biotin/avidin, magneticbeads (e.g. Dynabeads™), fluorescent dyes (e.g., fluoresceinisothiocyanate, texas red, rhodamine, green fluorescent protein, and thelike), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g.,horse radish peroxidase, alkaline phosphatase and others commonly usedin an ELISA), and colorimetric labels such as colloidal gold or coloredglass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads.

Means of detecting such labels are well known to those of skill in theart. Thus, for example, radiolabels may be detected using photographicfilm or scintillation counters, fluorescent markers may be detectedusing a photodetector to detect emitted illumination. Enzymatic labelsare typically detected by providing the enzyme with a substrate anddetecting the reaction product produced by the action of the enzyme onthe substrate, and calorimetric labels are detected by simplyvisualizing the colored label, and so forth.

C) Ligands.

As explained above, the effector molecule may also be a ligand or anantibody. Particularly preferred ligand and antibodies are those thatbind to surface markers on immune cells. Chimeric molecules utilizingsuch antibodies as effector molecules act as bifunctional linkersestablishing an association between the immune cells bearing bindingpartner for the ligand or antibody and the tumor cells overexpressingthe IL13 receptor. Suitable antibodies and growth factors are known tothose of skill in the art and include, but are not limited to, IL-2,IL-4, IL-6, IL-7, tumor necrosis factor (TNF), anti-Tac, TGFα, SEA, SEB,and the like.

D) Nucleic Acids

Nucleic acids can also be attached to the mutagenized IL13 molecules ofthis invention. In this context, the IL13 acts as a non-viral vectoreffectively delivering the nucleic acid to the target cell. The nucleicacids can be attached directly to the mutagenized IL13, or it can beattached through a linker or it can be complexed with or encapsulated inanother moiety (e.g., a lipid, a liposome, a viral coat, and the like)that is attached to the mutagenized IL13. The nucleic acid can provideany of a number of effector functions. The nucleic acid can encode oneor more proteins and thereby deliver a particular enzymatic activity,substrate and/or epitope to the cell. In this context, it is typicallydesirable to express the nucleic acid and the nucleic acid is preferablya component of an expression cassette. The expression cassette typicallyincludes a promoter initiation and termination codons and is selected tooptimize expression in the target cell. Methods of constructing suitableexpression cassettes are well known to those of skill in the art (see,e.g., Sambrook et al. (1989), Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory).

The nucleic acid may also have a particular activity in its own right.Thus the nucleic acid may be an anti-sense nucleic acid or a ribozymeselected in inhibit the expression of one or more target genes.

E) Sensitizing Agents.

Other effector molecules include sensitizing agents that render thetarget (e.g., tumor) cell susceptible to various cancer therapeutics.The sensitizing agent can be a drug or a gene (under the control of apromoter in an appropriate expression cassette to induce expression inthe target cell).

It has long been proposed that genes with a drug-conditional “killing”function be employed for treating tumors. For example, it has beenproposed that expression of the herpes simplex virus (HSV) thymidinekinase (TK) gene in proliferating cells, renders the cells sensitive tothe deoxynucleoside analog, ganciclovir (Moolten et at. (1986) CancerRes. 46:5276-5281; Moolten et al. (1990) Hum. Gene Ther. 1: 125-134;Moolten et al. (1990) J. Natl. Caucer Inst. 82: 297-300; Short et al.(1990) J. Neurosci Res. 27: 427-433; Ezzedine et al. (1991) New Biol. 3:608-614, Boviatsis et al. (1994) Hum. Gene Ther . 5: 183-191). HSV-TKmediates the phosphorylation of ganciclovir, which is incorporated intoDNA strands during DNA replication (S-phase) in the cell cycle, leadingto chain termination and cell death (Elion (1983) Antimicr. Chemother.12, sup. B:9-17).

A second example of a gene with a drug-conditional “killing” function isthe bacterial cytosine deaminase gene, which confers chemosensitivity tothe relatively non-toxic 5-fluorouracil precursor 5-fluorocytosine(Mullen et al. (1992) Proc. Natl. Acad. Sci USA 89: 33-37; Huber et al.(1993) Cancer Res. 53: 4619-4626; Mullen et al. (1994) Carcer Res. 54:1503-1506).

Still another example of a gene with a drug-conditional “killing”function is a cytochrome P450 gene. Expression, of the gene productrenders tumor cells sensitive to a chemotherapeutic agent, inparticular, cyclophosphamide or ifosphamide (see, U.S. Pat. No.5,688,773).

Other sensitizing agents need not be genes. Thus, for example, U.S. Pat.No. 4,282,233 describes compounds that treat multiple drug resistance ofsusceptible tumor cells. Use of the chimeric molecules of this inventionto deliver such compounds specifically to a tumor can reduce multipledrug resistance of the target cells rendering them susceptible toconventional cancer therapeutics.

F) Other Effector Moieties.

Other suitable effector molecules include pharmacological agents orencapsulation systems containing various pharmacological agents. Thus,the targeting molecule of the chimeric molecule may be attached directlyto a drug that is to be delivered directly to the tumor. Such drugs arewell known to those of skill in the art and include, but are not limitedto, doxorubicin, vinblastine, genistein, an antisense molecule, and thelike.

Alternatively, the effector molecule may be an encapsulation system,such as a liposome or micelle that contains a therapeutic compositionsuch as a drug, a nucleic acid (e.g. an antisense nucleic acid), oranother-therapeutic moiety that is preferably shielded from directexposure to the circulatory system. Means of preparing liposomesattached to antibodies are well known to those of skill in the art. See,for example, U.S. Pat. No. 4,957,735, Connor et al., Pharm. Ther., 28:341-365 (1985)

IV. Attachment of the Targeting Molecule to the Effector Molecule

One of skill will appreciate that the targeting molecule, mutagenizedIL13, and effector molecule(s) may be joined together in any order.Thus, the effector molecule may be joined to either the amino or carboxytermini of the mutagenized IL13. The mutagenized IL13 may also be joinedto an internal region of the effector molecule, or conversely, theeffector molecule may be joined to an internal location of themutagenized IL13, as long as the attachment does not interfere with therespective activities of the molecules.

The mutagenized IL13 and the effector molecule may be attached by any ofa number of means well known to those of skill in the art. Typically theeffector molecule is conjugated, either directly or through a linker(spacer), to the mutagenized IL13. However, where the effector moleculeis also a polypeptide it is preferable to recombinantly express thechimeric molecule as a single-chain fuision protein.

A) Conjugation of the Effector Molecule to the Mutagenized IL13.

In one embodiment, the targeting molecule (e.g., mutagenized IL13 orcircularly permuted mutagenized IL13) is chemically conjugated to theeffector molecule (e.g., a cytotoxin, a label, a ligand, or a drug orliposome). Means of chemically conjugating molecules are well known tothose of skill.

The procedure for attaching an agent (effector) to an IL13 will varyaccording to the chemical structure of the agent. Mutagenized IL13, likeother polypeptides, contains variety of functional groups; e.g.,carboxylic acid (COOH) or free amine (—NH₂) groups, which are availablefor reaction with a suitable functional group on an effector molecule tobind the effector thereto.

Alternatively, the mutagenized IL13 and/or effector molecule may bederivatized to expose or attach additional reactive functional groups.The derivatization may involve attachment of any of a number of linkermolecules such as those available from Pierce Chemical Company, RockfordIll.

A “linker”, as used herein, is a molecule that is used to join thetargeting molecule (mutagenized IL13) to the effector molecule. Thelinker is capable of forming covalent bonds to both the mutagenized IL13and to the effector molecule. Suitable linkers are well known to thoseof skill in the art and include, but are not limited to, straight orbranched-chain carbon linkers, heterocyclic carbon linkers, or peptidelinkers. Where the effector molecule is a polypeptides, the linkers maybe joined to the constituent amino acids through their side groups(e.g., through a disulfide linkage to cysteine). However, in a preferredembodiment, the linkers will be joined to the alpha carbon amino andcarboxyl groups of the terminal amino acids.

A bifunctional linker having one functional group reactive with a groupon a particular agent, and another group reactive with an antibody, maybe used to form the desired conjugate. Alternatively, derivatization mayinvolve chemical treatment of the mutagenized IL13. For example,procedures for generation of free sulffiydryl groups on polypeptide,such as antibodies or antibody fragments, are also known (See U.S. Pat.No. 4,659,839).

Many procedure and linker molecules for attachment of various compoundsincluding radionuclide metal chelates, toxins and drugs to proteins(e.g., to antibodies) are known. See, for example, European PatentApplication No. 188,256; U.S. Pat. Nos. 4,671,958, 4,659,839, 4,414,148,4,699,784; 4,680,338; 4,569,789; and 4,589,071; and Borlinghaus et al.Cancer Res. 47: 4071-4075 (1987). In particular, production of variousimmunotoxins is well-known within the art and can be found, for examplein “Monoclonal Antibody-Toxin Conjugates: Aiming the Magic Bullet,”Thorpe et al., Monoclonal Antibodies in Clinical Medicine, AcademicPress, pp. 168-190 (1982), Waldmann (1991) Science, 252: 1657, U.S. Pat.Nos. 4,545,985 and 4,894,443.

In some circumstances, it is desirable to free the effector moleculefrom the mutagenized IL13 when the chimeric molecule has reached itstarget site. Therefore, chimeric conjugates comprising linkages whichare cleavable in the vicinity of the target site may be used when theeffector is to be released at the target site. Cleaving of the linkageto release the agent from the mutagenized IL13 may be prompted byenzymatic activity or conditions to which the conjugate is subjectedeither inside the target cell or in the vicinity of the target site.When the target site is a tumor, a linker which is cleavable underconditions present at the tumor site (e.g. when exposed totumor-associated enzymes or acidic pH) may be used.

A number of different cleavable linkers are known to those of skill inthe art. See U.S. Pat. Nos. 4,618,492; 4,542,225, and 4,625,014. Themechanisms for release of an agent from these linker groups include, forexample, irradiation of a photolabile bond and acid-catalyzedhydrolysis. U.S. Pat. No. 4,671,958, for example, includes a descriptionof immunoconjugates comprising linkers which are cleaved at the targetsite in vivo by the proteolytic enzymes of the patient's complementsystem. In view of the large number of methods that have been reportedfor attaching a variety of radiodiagnostic compounds, radiotherapeuticcompounds, drugs, toxins, and other agents to antibodies one skilled inthe art will be able to determine a suitable method for attaching agiven agent to an antibody or other polypeptide.

B) Production of Mutagnized IL13 Fusion Proteins.

Where the effector molecule is a polypeptide, the chimeric mutagenizedIL13-effector molecules can be prepared as fusion proteins. The fusionproteins can be chemically synthesized as described above formutagenized IL13.

However, in a preferred embodiment, the chimeric fusion proteins of thepresent invention are synthesized using recombinant DNA methodology.Generally this involves creating a DNA sequence that encodes the fuisionprotein, placing the DNA in an expression cassette under the control ofa particular promoter, expressing the protein in a host, isolating theexpressed protein and, if required, renaturing the protein.

DNA encoding the fuision proteins (e.g. IL13.E13K-PE38QQR) of thisinvention may be prepared by any suitable method, including, forexample, cloning and restriction of appropriate sequences or directchemical synthesis by methods such as the phosphotriester method ofNarang et al. (1979) Meth. Enzymol. 68: 90-99; the phosphodiester methodof Brown et al. (1979) Meth. Enzymol. 68: 109-151; thediethylphosphoramidite method of Beaucage et al. (1981) Tetra. Lett.,22: 1859-1862; and the solid support method of U.S. Pat. No. 4,458,066.

Chemical synthesis produces a single stranded oligonucleotide. This maybe converted into double stranded DNA by hybridization with acomplementary sequence, or by polymerization with a DNA polymerase usingthe single strand as a template. One of skill would recognize that whilechemical synthesis of DNA is limited to sequences of about 100 bases,longer sequences may be obtained by the ligation of shorter sequences.

Alternatively, subsequences may be cloned and the appropriatesubsequences cleaved using appropriate restriction enzymes. Thefragments may then be ligated to produce the desired DNA sequence.

In a preferred embodiment, DNA encoding fusion proteins of the presentinvention may be cloned using DNA amplification methods such aspolymerase chain reaction (PCR). Thus, in a preferred embodiment, thegene for IL13 is PCR amplified, using primers that introduce one or moremutations. The primers preferably include restrictions sites, e.g., asense primer containing the restriction site for NdeI and an antisenseprimer containing the restriction site for HindIII. In a particularlypreferred embodiment, the primers are selected to amplify the nucleicacid starting at position 19, as described by McKenzie et al. (1987),supra. This produces a nucleic acid encoding the mature IL13 sequenceand having terminal restriction sites. A PE38QQR fragment may be cut outof the plasmid pWDMH4-38QQR or plasmid pSGC242FdN1 described by Debinskiet al. Int. J Cancer, 58: 744-748 (1994), and by Debinski et al. (1994)Clin. Cancer Res., 1: 1015-1022 respectively. Ligation of themutagenized IL13 and a Pseudomonas exotoxin (e.g., PE38QQR) sequencesand insertion into a vector produces a vector encoding the mutagenizedIL13 joined to the terminus of the Pseudomonas exotoxin (e.g., joined tothe amino terminus of PE38QQR or PEE (position 253 of the PE)). In apreferred embodiment, the two molecules are joined directly.Alternatively there can be an intervening peptide linker (e.g.,. a threeamino acid junction consisting of glutamic acid, alanine, andphenylalanine introduced by the restriction site).

While the two molecules are preferably essentially directly joinedtogether, one of skill will appreciate that the molecules may beseparated by a peptide spacer consisting of one or more amino acids.Generally the spacer will have no specific biological activity otherthan to join the proteins or to preserve some minimum distance or otherspatial relationship between them. However, the constituent amino acidsof the spacer may be selected to influence some property of the moleculesuch as the solubility, folding, net charge, or hydrophobicity.

The nucleic acid sequences encoding the fusion proteins may be expressedin a variety of host cells, including E. coli, other bacterial hosts,yeast, and various higher eukaryotic cells such as the COS, CHO and HeLacells lines and myeloma cell lines. The recombinant protein gene will beoperably linked to appropriate expression control sequences for eachhost. For E. coli this includes a promoter such as the T7, trp, orlambda promoters, a ribosome binding site and preferably a transcriptiontermination signal. For eukaryotic cells, the control sequences willinclude a promoter and preferably an enhancer derived fromimmunoglobulin genes, SV40, cytomegalovirus, etc., and a polyadenylationsequence, and may include splice donor and acceptor sequences.

The plasmids of the invention can be transferred into the chosen hostcell by well-known methods such as calcium chloride, or heat shock,transformation for E. coli and calcium phosphate treatment orelectroporation for mammalian cells. Cells transformed by the plasmidscan be selected by resistance to antibiotics conferred by genescontained on the plasmids, such as the amp, gpt, neo and hyg genes.

Once expressed, the recombinant fusion proteins can be purifiedaccording to standard procedures of the art, including ammonium sulfateprecipitation, affinity columns, column chromatography, gelelectrophoresis and the like (see, generally, R. Scopes, ProteinPurification, Springer-Verlag, N.Y. (1982), Deutscher, Methods inEnzymology VoL. 182: Guide to Protein Punification., Academic Press,Inc. N.Y. (1990)). Substantially pure compositions of at least about 90to 95% homogeneity are preferred, and 98 to 99% or more homogeneity aremost preferred for pharmaceutical uses. Once purified, partially or tohomogeneity as desired, the polypeptides may then be usedtherapeutically.

One of skill in the art would recognize that after chemical synthesis,biological expression, or purification, the IL13 receptor targetedfusion protein may possess a conformation substantially different thanthe native conformations of the constituent polypeptides. In this case,it may be necessary to denature and reduce the polypeptide and then tocause the polypeptide to re-fold into the preferred conformation.Methods of reducing and denaturing proteins and inducing re-folding arewell known to those of skill in the art (See, Debinski et al. (1993) J.Biol. Chem., 268: 14065-14070; Kreitman and Pastan (1993) Bioconjug.Chem., 4: 581-585; and Buchner, et al. (1992) Anal. Biochem., 205:263-270). Debinski et al., for example, describe the denaturation andreduction of inclusion body proteins in guanidine-DTE. The protein isthen refolded in a redox buffer containing oxidized glutathione andL-arginine.

One of skill would recognize that modifications can be made to the IL13receptor targeted fusion proteins without diminishing their biologicalactivity. Some modifications may be made to facilitate the cloning,expression, or incorporation of the targeting molecule into a fusionprotein. Such modifications are well known to those of skill in the artand include, for example, a methionine added at the amino terminus toprovide an initiation site, or additional amino acids placed on eitherterminus to create conveniently located restriction sites or terminationcodons.

V. Identificatlon of Target Cells

The mutagenized IL13 molecules of this invention are particularly wellsuited as targeting moieties for binding tumor cells because tumorcells, overexpress IL13 receptors. In particular, carcinoma tumor cells(e.g. renal carcinoma cells) overexpress IL13 receptors at levelsranging from about 2100 sites/cell to greater than 150,000 sites percell. Similarly, gliomas and Kaposi's sarcoma also overexpress IL13receptors (IL13R). Moreover, substantially every cancer type tested todate appears to overexpress IL13 receptors as compared to thecorresponding “healthy” tissue. Thus it appears that IL13 receptoroverexpression is general characteristic of a neoplastic cells.

Thus, the methods of this invention can be used to target an effectormolecule to virtually any neoplastic cell. Neoplasias are well known tothose of skill in the art and include, but are not limited to, cancersof the skin (e.g., basal or squarnous cell carcinoma, melanoma, Kaposi'ssarcoma, etc.), cancers of the reproductive system (e.g., testicular,ovarian, cervical), cancers of the gastrointestinal tract (e.g.,stomach, small intestine, large intestine, colorectal, etc.), cancers ofthe mouth and throat (e.g. esophageal, larynx, oropharynx, nasopharynx,oral, etc.), cancers of the head and neck, bone cancers, breast cancers,liver cancers, prostate cancers (e.g., prostate carcinoma), thyroidcancers, heart cancers, retinal cancers (e.g., melanoma), kidneycancers, lung cancers (e.g., mesothelioma), pancreatic cancers, braincancers (e.g. gliomas, medulloblastomas, meningiomas, etc.) and cancersof the lymph system (e.g. lymphoma).

In a particularly preferred embodiment, the methods of this inventionare used to target effector molecules to kidney cancers, to skin cancers(especially Kaposi's sarcoma), and to brain cancers (especially gliomas,and medulloblastomas).

One of skill in the art will appreciate that identification andconfirmation of IL13 overexpression by other cells requires only routinescreening using well-known methods. Typically this involves providing alabeled molecule that specifically binds to the IL13 receptor (e.g., anative or mutagenized IL13). The cells in question are then contactedwith this molecule and washed. Quantification of the amount of labelremaining associated with the test cell provides a measure of the amountof IL13 receptor (IL13R) present on the surface of that cell. In apreferred embodiment, IL13 receptor may be quantified by measuring thebinding of ¹²⁵I-labeled IL13 (¹²⁵I-IL13) to the cell in question.Details of such a binding assay are provided in U.S. Pat. No. 5,614,191.

VI. Pharmaceutical Compositions

The mutagenized IL13 chimeric molecules (e.g., hIL13.E13K-PE4E and othercytotoxins) of this invention are useful for parenteral, topical, oral,or local administration, such as by aerosol or transdermally, forprophylactic and/or therapeutic treatment. The pharmaceuticalcompositions can be administered in a variety of unit dosage formsdepending upon the method of administration. For example, unit dosageforms suitable for oral administration include powder, tablets, pills,capsules and lozenges. It is recognized that the fusion proteins andpharmaceutical compositions of this invention, when administered orally,must be protected from digestion. This is typically accomplished eitherby complexing the protein with a composition to render it resistant toacidic and enzymatic hydrolysis or by packaging the protein in anappropriately resistant carrier such as a liposome. Means of protectingcompounds from digestion are well known in the art (see, e.g., U.S. Pat.No. 5,391,377 describing lipid compositions for oral delivery oftherapeutic agents).

The pharmaceutical compositions of this invention are particularlyuseful for parenteral administration, such as intravenous administrationor administration into a body cavity or lumen of an organ. Thecompositions for administration will commonly comprise a solution of thechimeric molecule dissolved in a pharmaceutically acceptable carrier,preferably an aqueous carrier. A variety of aqueous carriers can beused, e.g. buffered saline and the like. These solutions are sterile andgenerally free of undesirable matter. These compositions may besterilized by conventional, well known sterilization techniques. Thecompositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, toxicity adjusting agents and thelike, for example, sodium acetate, sodium chloride, potassium chloride,calcium chloride, sodium lactate and the like. The concentration ofchimeric molecule in these formulations can vary widely, and will beselected primarily based on fluid volumes, viscosities, body weight andthe like in accordance with the particular mode of administrationselected and the patient's needs.

Thus, a typical pharmaceutical composition for intravenousadministration would be about 0.1 to 10 mg per patient per day. Dosagesfrom 0.1 up to about 100 mg per patient per day may be used,particularly when the drug is administered to a secluded site and notinto the blood stream, such as into a body cavity or into a lumen of anorgan. Typically dosages will be adjusted to maximize dose whilemaintaining adverse effects at generally acceptable levels. Actualmethods for preparing parenterally administrable compositions will beknown or apparent to those skilled in the art and are described in moredetail in such publications as Remington's Pharmaceutical Science, 15thed., Mack Publishing Company, Easton, Pa. (1980).

The compositions containing the present chimeric molecules (e.g., fusionproteins), or a cocktail thereof (i.e., with other proteins, e.g.,TGFα-PE38QQR), can be administered for therapeutic treatments. Intherapeutic applications, compositions are administered to a patientsuffering from a disease, in an amount sufficient to cure or at leastpartially arrest the disease and its complications. An amount adequateto accomplish this is defined as a “therapeutically effective dose.”Amounts effective for this use will depend upon the severity of thedisease and the general state of the patient's health.

Single or multiple administrations of the compositions may beadministered depending on the dosage and frequency as required andtolerated by the patient. In any event, the composition should provide asufficient quantity of the proteins of this invention to effectivelytreat the patient.

Among various uses of the cytotoxic fuision proteins of the presentinvention are included a variety of disease conditions caused byspecific human cells that may be eliminated by the toxic action of theprotein. One preferred application is the treatment of cancer (e.g., aglioma), such as by the use of an mutagenized IL13 ligand attached to acytotoxin (e.g., PE or a PE derivative).

It will be appreciated by one of skill in the art that there are someregions that are not heavily vascularized or that are protected by cellsjoined by tight junctions and/or active transport mechanisms whichreduce or prevent the entry of macromolecules present in the bloodstream. Thus, for example, systemic administration of therapeutics totreat gliomas, or other brain cancers, is constrained by the blood-brainbarrier which resists the entry of macromolecules into the subarachnoidspace.

One of skill in the art will appreciate that in these instances, thetherapeutic compositions of this invention can be administered directlyto the tumor site. Thus, for example, brain tumors (e.g., gliomas) canbe treated by administering the therapeutic composition directly to thetumor site (e.g., through a surgically implanted catheter). Where thefluid delivery through the catheter is pressurized, small molecules(e.g. the therapeutic molecules of this invention) will typicallyinfiltrate as much as two to three centimeters beyond the tumor margin.

Alternatively, the therapeutic composition can be placed at the targetsite in a slow release formulation (e.g., a thrombin-fibrinogenmixture). Such formulations can include, for example, a biocompatiblesponge or other inert or resorbable matrix material impregnated with thetherapeutic composition, slow dissolving time release capsules ormicrocapsules, and the like.

Typically the catheter, or catheters, or time release formulation willbe placed at the tumor site as part of a surgical procedure. Thus, forexample, where major tumor mass is surgically debulked, the perfusingcatheter or time release formulation can be emplaced at the tumor siteas an adjunct therapy. Of course, surgical removal of the tumor mass maybe undesired, not required, or impossible, in which case, the deliveryof the therapeutic compositions of this invention may comprise theprimary therapeutic modality.

VII. Diagnostic Kits

In another embodiment, this invention provides for kits for thetreatment of tumors or for the detection of cells overexpressing IL13receptors. Kits will typically comprise a chimeric molecule of thepresent invention (e.g. mutagenized IL13-label, mutagenizedIL13-cytotoxin, mutagenized IL13-ligand, etc.). In addition the kitswill typically include instructional materials disclosing means of useof chimeric molecule (e.g. as a cytotoxin, for detection of tumor cells,to augment an immune response, etc.). The kits may also includeadditional components to facilitate the particular application for whichthe kit is designed. Thus, for example, where a kit contains a chimericmolecule in which the effector molecule is a detectable label, the kitmay additionally contain means of detecting the label (e.g. enzymesubstrates for enzymatic labels, filter sets to detect fluorescentlabels, appropriate secondary labels such as a sheep anti- mouse-HRP, orthe like). The kits may additionally include buffers and other reagentsroutinely used for the practice of a particular method. Such kits andappropriate contents are well known to those of skill in the art.

EXAMPLES

The following examples are offered to illustrate, but not to limit thepresent invention.

Recently, we have demonstrated that the vast majority of brain cancers(gliomas) abundantly express a receptor (R) for interleukin 13 (IL13).In order to achieve even more specific targeting of the IL13R ingliomas, we have mutagenized human (h) IL13. The mutation was made toalter IL13 interaction with the shared functional IL13/IL4 normal tissuereceptor, but not with the glioma-associated receptor.

In one embodiment this invention involved mutating glutamic acid atposition 13 to lysine. This mutant, designated hIL13.E13K was fused itto derivatives of Pseudomonas exotoxin A (PE). As demonstrated in thefollowing examples, the mutated IL13-based cytotoxins are less active onnormal cells and less toxic in vivo, and are better anti-tumor agentswhen compared with the cytotoxins containing non-mutagenized hIL13. Weconclude that hIL13 is amenable to engineering which leads to a morediscriminate recognition of the hIL13R that is cancer-associated fromthe shared IL13/IL4 R of normal tissue.

Example 1 Creation of Mutagenized IL13 and IL13 Cytotoxins

Materials

Restriction endonucleases and DNA ligase were obtained from New EnglandBiolabs (Beverly, Mass.), Bethesda Research Laboratories (BRL,Gaithersburg, Md.) and Boehringer Mannheim (Indianapolis, Ind.). Fastprotein liquid chromatography (FPLC) columns and media wore purchasedfrom Pharmacia (Piscataway, N.J.). Oligonucleotide primers weresynthesized at Macromolecular Core Laboratory at the Penn State Collegeof Medicine. PCR kit was from Perkin-Elmer Cetus (Norwalk, Conn.).MTS/PMS for cell titer 96 aqueous non-radioactive cell proliferationassay was purchased from Promega (Madison, Wis.).

Plasmids, Bacterial Strains and Cell Lines.

Plasmids carry a T7 bacteriophage late promoter, a T7 transcriptionterminator at the end of the open reading frame of the protein, a f1origin of replication and a gene for ampicillin resistance (Debinski etal. (1992) J. Clin. Invest. 90: 405-411). The cDNA encoding sequence forhIL13 was PCR-cloned to produce hIL13-PE38QQR, hIL13-PE4E, and hIL13, asdescribed previously (Debinski et al. (1995) Clin. Cancer Res. 1:1253-1258; Debinski et al. (1996) J. Biol. Chem. 271: 22428-22433;Debinski et al. (1995) J. Biol. Chem. 270: 16775-16780). Recombinantproteins were expressed in E. coli BL21 (λDE3) under control of the T7late promoter (Studier et al. (1986) J. Mol. Biol. 189: 113-130).Plasmids were amplified in E. coli (HB101 or DH5α high efficiencytransformation) (BRL) and DNA was extracted using Qiagen kits(Chatsworth, Calif.).

Construction of Plasmids Encoding hIL13.E13K-cytotoxins

Human IL13 (hIL3) was PCR-amplified in its mature form from a phuIL13template (Debinski et al. (1995) J. Biol. Chem. 270: 16775-16780). Amutation of glutamic acid to lysine at position 13 in human interleukin13 was incorporated into the sense PCR primer (hIL13.E13K sense primer5′-AGGAGATATACATATGTCCCCAGGCCCTGT GCCTCCCTCTACAGCCCTCAGGAAGCTCATTGAGGA-3′, SEQ ID NO: 1). The antisense primer was5′-GTCGTGGGTGGATCCTCAGTTGAACCGTCCCTCGCGAA-3′ (SEQ ID NO: 2).

New sites were introduced for two restriction endonucleases Nde I andHind III at the 5′and 3′ends of the interleukin gene, respectively. Thedigested fragments were subcloned into the expression vectors digestedwith the appropriate enzymes. The plasmids were sequenced using anautomated ABI PRISM™ 377 DNA Sequencer (Foster City, Calif.) in theCollege of Medicine Molecular Genetics Core Facility.

Expression and Purification of Recombinant Proteins

E. coli BL21 (λDE3) cells were transformed with plasmids of interest andcultured in Terrific broth (Gibco, BRL). The cytotoxins and interleukinswere localized to the inclusion bodies. The procedure for therecombinant proteins isolation from the inclusion bodies was describedpreviously (Debinski et al. (1993) J. Biol. Chem. 268: 14065-14070).After dialysis, the renatured proteins were purified on ion-exchangecolumns and by size exclusion chromatography on Sephacryl S-100HR(Pharmacia). Protein concentration was determined by the Bradford assay(Pierce “Plus”, Rocklford, Ill.) using BSA as a standard.

The recombinant proteins were expressed and purified to a high level ofhomogeneity as determined by STS-polyacrylamide gel electrophoresis.Electrophoresis was performed using 15% reduced SDS polyacrylamide gelstained with Coomassie Blue. Sephacryl S-100 HR purified recombinantproteins were loaded at 6 μg/lane. hIL13/IL13.E13K,hIL13-PE38QQR/hIL13.E13K-PE38QQR, hIL13-PE4E/hIL13.EPE4E were 12-, 50-,and 78-kDa proteins, respectively.

Example 2 Activity of Mutagenized IL13 Chimeric Molecules

Methods

Protein Synthesis Inhibition Assay

The activities of cytotoxins were tested as follows: usually 5×10³ cellsper well were plated in a 96-well tissue culture plate in 150 μl ofmedia. Various concentrations of cytotoxins were diluted in 0.1% BSA/PBSand 25 μl of each dilution was added to cells 18-24 h following cellplating. Cells were incubated at 37° C. for another 48 h. Then, thecytotoxicity was determined using a calorimetric MTS[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, innersalt]/PMS (phenazine methasulfate) cell proliferation assay. MTS/PMS wasadded at a half final concentration as recommended by the manufacturer.The cells were incubated with the dye for 4 hr and then the absorbancewas measured at 490 nnm for each well using a micro-plate reader(Cambridge Technology, Inc., Watertown, Mass.). The wells containingcells treated with cycloheximide (10 mM) or wells with no viable cellsleft served as a background for the assay. We have used the samesolutions of the cytotoxins for concomitant studies on normal and cancercells. For blocking studies, interleukins at a concentration of 1.0μg/ml were added to cells for 60 min before the cytotoxins addition. Theresults are expressed as IC₅₀ which is a concentration at which thecytotoxin inhibits the protein synthesis by 50%.

Cell Proliferation Studies

Cell proliferation studies using TF-1 cells were performed essentiallyas described (Zurawski et al. (1993) EMBO J. 12: 2663-2670). Maximalproliferative activities of the interleukins were obtained at aconcentration of 100 ng/ml. The values were expressed as the differencebetween the background and maximal (recombinant proteins treated cells)MTS conversion that was recorded at A₄₉₀ nm. Data were obtained from theaverage of quadruplicates and the assays were repeated at least fourtimes.

Binding Experiments of Recombinant Interleukins and their Cytotoxins toHuman Glioma Cells

1 ×10⁶ U-251 MG glioma cells was incubated with 100 μM ¹²⁵I-hIL13 aloneor in the presence of 0.1-500 nM unlabeled hIL13.E13K and hIL13 orhIL13.E13K-PE4E and hIL13-PE4E in duplicate tubes. The incubation wascarried out at 4° C. for 4 hrs. The cultures were centrifuged (10,000×g) through a phthalate oil mixture at 4° C. to separate cell-bound¹²⁵I-hIL13 from free ¹²⁵I-hIL13. The cell pellet was cut off from eachtube and radioactivity counted in a gamma counter. The experiment wasdone twice.

Anti-tumor Experiments

The U251-MG human malignant glioma cells (6×10⁶ per mouse) wereimplanted subcutaneously into female nu/nu athymic mice (4 to 5-wk old)on day 0. The treatment. started on day 11 or 12 when established tumorswere formed. Cytotoxins were injected intratumorally as this route ofdrug delivery is envisioned for future clinical trials with hIL13-basedcytotoxins (Wersall et al. (1997) Cancer Immunol. Immunother. 44:157-164; Laske et al. (1997). Nature Medicine 3: 1362-1368). Theinjection volume was 25 μl and cytotoxins were diluted in PBS/0.1% BSA.Each treatment group was composed of five animals. Tumors were measuredwith a caliper and the formula for tumor volume calculation was asfollows: length×width²×0.4 (Debinski et al. (1993) J. Biol. Chem. 268:14065-14070).

RESULTS

hIL13.E13K Proliferative Activity is Altered

We have measured proliferative responses to interleukins and theirmutants in TF-1 cells (a human pre-myeloid erythrocytic leukemic cellswhich do express the shared IL13/4R) (Obiri et al. (1997) J. Immunol.158: 756-764; Zurawski et al. (1993) EMBO J. 12: 2663-2670). We havetreated TF-1 cells with hIL13, hIL13.E13K, and hIL4.Y124D (FIG. 4).hIL13 was very potent in stimulating the growth of TF-1 cells (FIG. 4).In contrast, hIL13.E13K was very weakly active, while hIL4.Y124D did notshow any proliferative activity on its own on these cells.

hIL13.E13K Fused to a Bacterial Toxin is Less Active on Normal HumanCells than a Wild Type hIL13-containing Cytotoxin

We next used normal human unbilical vein endothelial cells (HUVEC) whichdo express functional hIL13/4R as other selective normal tissues(Schnyder et al. (1996) Blood 87: 4286-4295, Bochner et al. (1995) J.Immunol. 154: 799-803, Sironi (1994) Blood 84: 1913-1921). We have foundno IC₅₀ for hIL13-PE38QQR on these cells at up to 10,000 ng/mlconcentration of the cytotoxin (Husain et al. (1997) Clin. Cancer Res.3: 151-156). However, the more potent cytotoxin on cancer cells invitro, hIL13-PE4E (Debinski et al. (1996) J. Biol. Chem. 271:22428-22433), showed some killing activity on HUVEC (IC₅₀ of 200-400ng/ml) (FIG. 5). When assayed side by side, hIL13.E13K-PE4E was fivetimes less cytotoxic to HUVEC (IC₅₀ of˜1000 ng/ml) than the cytotoxincontaining a wild type hIL13. It is important to emphasize the need forhigh concentrations of the cytotoxins to evoke any effect on normalcells.

hIL13.E13K Cytotoxins Gain in a Potency on Human Glioma Cells

Since hIL13.E13K-PE4E showed a lesser cytotoxicity than hIL13-PE4E onnormal cells (FIG. 5) it was possible that it may lose its potency oncancer cells as well. Thus, we treated several glioma cell lines withhIL13 cytotoxins. We have found that, e.g., SNB-19 glioma cells areextremely sensitive to hIL13.E13K-PE4E at an IC₅₀ as low as 0.7 pg/ml(FIG. 6A). On the other hand, although very potent, the cytotoxic actionof hIL13-PE4E was surprisingly six times less than the one seen with thehIL13.E13K-PE4E (FIG. 6A). Similar gain in the cytotoxic potencyofhIL13.E13K-PE4E over hIL13-PE4E was observed on U-251 MG cells (FIG.6B). On average, several other glioma cells were killed 3 to 10 timesmore potently with hIL13.E13K-PE4E than with hIL13-PE4E. Theseunanticipated results are in sharp contrast to a decrease in thecytotoxic activity of hIL13.E13K-PE4E on normal cells (FIG. 5).

hIL13 and hIL13.E13K. but not hIL4. Block the Action of hIL13.E13KCytotoxins on Glioma Cells

We also pretreated glioma cells with either hIL13 or hIL13.E13K beforethe addition of the cytotoxic fusion proteins. We found that thecytotoxic action of hIL13.E13K-PE4E is hIL13R-specific since it isblocked by an excess of hIL13 on all tested glioma cells (e.g., DBTRG MGin FIG. 6C). Moreover, hIL13.E13K neutralized the cytotoxicity ofhIL13.E13K-PE4E similarly to hIL13. In contrast, but in accord with ourprevious studies (Debinski et al. (1995) Clin. Cancer Res. 1: 1253-1258;Debinski etal. (1996) J. Biol. Chem. 271: 22428-22433) hIL4 wasineffective in neutralizing the action of hIL13.E13K-PE4E (FIG. 6C).

Binding Avidity of Mutated hIL13 to Glioma Cells

To determine a possible reason for an unexpected enhanced potency ofhIL13.E13K cytotoxins, the hIL13.E13K mutant and hIL13 were used in acompetition assay for the binding sites for ¹²⁵I-hIL13 on U-251 MGcells. The representative results are shown in FIG. 7. Both the mutatedand wild type hIL13 competed efficiently for the radiolabeled hIL13binding sites (FIG. 7). However, hIL13.E13K was approximately 50-foldbetter binding molecule than hIL13. Also hiL13.E13K-PE4E showed 8 to 10times better affinity to glioma cells than hIL13-PE4E (data not shown).

Anti-tumor Activities of hIL3 Cytotoxins

Since we have tested the interaction of cytotoxins containingmutagenized hIL13 with brain tumor cells in vitro (FIG. 6), it wasimportant to compare their effects with that of wild typeinterleukin-containing cytotoxins in vivo. We have performed anti-tumorexperiments in mice bearing the U-251 MG xenografis of human malignantglioma. IL13 is not species-specific therefore animal studies mayclosely reflect human situation. The treatment started on day 12 whentumors were formed and were larger than 50 mm³ (FIG. 8A). Threeinjections of hIL13.E13K-PE4E every other day produced completeregressions in groups of mice receiving 4 μg and 0.5 μg per mouse of thecytotoxin, while 0.0625 μg of hIL13.E13K-PE4E per mouse evoked tumorgrowth inhibition. There were two deaths in the group receiving 4.0 μgof the cytotoxin but surviving mice remained free of tumor. Three out offive mice that received 0.5 μg of hIL13.E13K-PE4E per mouse were free oftumor on day 80. On the other hand, neither 2.0 μg per mouse nor 0.25 μgper mouse of hIL13-PE4E resulted in complete regressions in all treatedmice, and there were 3 deaths in the former group of mice (FIG. 8A). Ofinterest, the tumor sites in the groups injected with hIL13.E13K-basedcytotoxin appeared distinctively more inflammatory when compared withthose in hIL13-PE4E-injected mice that even resulted in skin wounds. Ina pilot experiment, we also found that six injections of 0.5 μg ofhIL13.E13K-PE4E per mouse produced cures (100 days free of tumor) in alltreated animals. The optimization of the treatment using hIL13.E13Kcytotoxins is underway.

In a subsequent experiment, we used hIL13.E13K-PE388QQR andhIL13-PE388QQR which are less active than PE4E-containing cytotoxins oncancer cells in vitro (FIG. 8B). The mice bearing established U-251 MGtumors were treated with six injections of 5.0, 1.0, and 0.2 μg ofeither cytotoxin per mouse every other day (FIG. 8B). All mice treatedwith 5.0 μg of hIL13-PE38QQR per mouse were dead after the 4th injectionwhile three out five mice survived this regimen in thehIL13.E13K-PE38QQR-treated group of animals (FIG. 8B). Tumors regressedin all mice treated with 1.0 μg of hIL13.E13K-PE38QQR per mouse whilethe same dose of hIL13-PE38QQR caused an arrest of tumors growth.Similarly, 0.2 μg of hIL13.E13K-PE38QQR per mouse produced much betteranti-tumor effect than the corresponding dose of 0.2 μg of hIL13-PE38QQRper mouse (FIG. 8B). Again, the tumor sites in hIL13.E13K-basedcytotoxin treated mice were visibly more inflammatory when compared withthe hIL13-based cytotoxin treated mice, particularly at higher doses ofthe cytotoxins.

DISCUSSION

We have found that mutagenized hIL13, hIL13.E13K, in a cytotoxin is lesstoxic and exhibits better anti-tumor activity than the fusion.proteinsbased on the wild type interleukin. These data indicate an effective wayto amplify therapeutic specificity of a tumor-associated receptor.

The functional normal tissue receptor for hIL13 interacts with hIL4because it involves the 140-kDa hIL4R_(β)(Obiri et al. (1997) J.Immunol. 158: 756-764, Zurawski et al. (1993) EMBO J. 12: 2663-2670).The change in a conservative amino acid that has been implicated in thebinding of hIL4 to the 140-kDa IL4R_(β)(Kruse et al. (1992) EMBO J. 12:5121-5129) has caused alteration to the behavior of a mutagenized hIL13as well. This further suggests that hIL13 is a structural homologue ofhIL4. Furthermore, it is plausible that the interaction between hIL13and the 140-kDa IL4R_(β) is, at least in part, through a directassociation of the hIL13 receptor binding region, located in its α-helixA, with the extracellular domain of the 140-kDa IL4R_(β). Furtherstudies are underway to address these specific issues.

It is intriguing that the hIL13.E13K-containing cytotoxins are betteranti-tumor agents while being less cytotoxic to normal cells and lesstoxic in vivo. It appears that hIL13.E13K may bind to glioma cells moreavidly than the wild type interleukin and by this contributing to abetter potency of cytotoxins. An increase in affinity may be areflection of the shift in the binding of hIL13.E13K to a particularform(s) of the hIL13R that is present on cancer cells (Murata et al.(1997) Biochem. Biophys. Res. Comm. 238: 92-94). Also, we have observeddifferences in gross morphology of the tumor sites treated withhIL13.E13K-based cytotoxins. This is compatible with a more vigorousinflammatory infiltration into the region of dying cancer cells notbeing altered when hIL13.E13K cytotoxins are used, or, this phenomenonis related to a faster and more potent cancer cells killing.

The search for more active targeted cytotoxins has been directed at theimprovement of their binding to the targeted receptor or betterutilization of the pathways involved in the bacterial toxins' processingand/or intra-cytosolic delivery (Seetharam et al. (1991) J. Biol. Chem.266: 17376-17381, Kreitman et al. (1994) Proc. Natl. Acad. Sci. 91:6889-6893). These improvements affect normal cells as the cytotoxinsbecome more toxic to them. Also, attempts at increasing efficacy oftraditional anti-cancer drugs, chemotherapeutics, are linked to theirmore severe toxicities and more prominent mutagenicity (Chinnasamy etal. (1997) Blood 89: 1566-1573). Our approach, on the other hand, wasfirst to decrease the already low interaction of cytotoxins with normalcells. We have identified a system in which this does not have to beparalleled by a decrease in the activity on brain tumor cells.

In sumnary, we have documented a unique single-reagent approach toincrease the specificity of cytotoxins that are targeted to aglioma-associated receptor. This includes their diminished intracellularsignaling on normal cells, an impaired targeting to normal cells, and anincreased in anti-tumor activity on brain tumors.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

5 1 65 DNA Artificial Sequence misc_feature PCR primer for amplifyinghuman IL-13 gene 1 aggagatata catatgtccc aggccctgtg cctccctctacagccctcag gaagctcatt 60 gagga 65 2 38 DNA Artificial Sequencemisc_feature PCR primer for amplifying human IL-13 gene 2 gtcgtgggtggatcctcagt tgaaccgtcc ctcgcgaa 38 3 132 PRT homo sapiens 3 Met Ala LeuLeu Leu Thr Thr Val Ile Ala Leu Thr Cys Leu Gly Gly 1 5 10 15 Phe AlaSer Pro Gly Pro Val Pro Pro Ser Thr Ala Leu Arg Glu Leu 20 25 30 Ile GluGlu Leu Val Asn Ile Thr Gln Asn Gln Lys Ala Pro Leu Cys 35 40 45 Asn GlySer Met Val Trp Ser Ile Asn Leu Thr Ala Gly Met Tyr Cys 50 55 60 Ala AlaLeu Glu Ser Leu Ile Asn Val Ser Gly Cys Ser Ala Ile Glu 65 70 75 80 LysThr Gln Arg Met Leu Ser Gly Phe Cys Pro His Lys Val Ser Ala 85 90 95 GlyGln Phe Ser Ser Leu His Val Arg Asp Thr Lys Ile Glu Val Ala 100 105 110Gln Phe Val Lys Asp Leu Leu Leu His Leu Lys Lys Leu Phe Arg Glu 115 120125 Gly Arg Phe Asn 130 4 114 PRT homo sapiens 4 Ser Pro Gly Pro Val ProPro Ser Thr Ala Leu Arg Glu Leu Ile Glu 1 5 10 15 Glu Leu Val Asn IleThr Gln Asn Gln Lys Ala Pro Leu Cys Asn Gly 20 25 30 Ser Met Val Trp SerIle Asn Leu Thr Ala Gly Met Tyr Cys Ala Ala 35 40 45 Leu Glu Ser Leu IleAsn Val Ser Gly Cys Ser Ala Ile Glu Lys Thr 50 55 60 Gln Arg Met Leu SerGly Phe Cys Pro His Lys Val Ser Ala Gly Gln 65 70 75 80 Phe Ser Ser LeuHis Val Arg Asp Thr Lys Ile Glu Val Ala Gln Phe 85 90 95 Val Lys Asp LeuLeu Leu His Leu Lys Lys Leu Phe Arg Glu Gly Arg 100 105 110 Phe Asn 5114 PRT homo sapiens 5 Ser Pro Gly Pro Val Pro Pro Ser Thr Ala Leu ArgLys Leu Ile Glu 1 5 10 15 Glu Leu Val Asn Ile Thr Gln Asn Gln Lys AlaPro Leu Cys Asn Gly 20 25 30 Ser Met Val Trp Ser Ile Asn Leu Thr Ala GlyMet Tyr Cys Ala Ala 35 40 45 Leu Glu Ser Leu Ile Asn Val Ser Gly Cys SerAla Ile Glu Lys Thr 50 55 60 Gln Arg Met Leu Ser Gly Phe Cys Pro His LysVal Ser Ala Gly Gln 65 70 75 80 Phe Ser Ser Leu His Val Arg Asp Thr LysIle Glu Val Ala Gln Phe 85 90 95 Val Lys Asp Leu Leu Leu His Leu Lys LysLeu Phe Arg Glu Gly Arg 100 105 110 Phe Asn

What is claimed is:
 1. A chimeric molecule having the formula:R¹—(L)_(j)—(R²)_(n) wherein R¹ is a polypeptide comprising the aminoacid sequence of SEQ ID NO:5; j is 0 or 1; n is at least 1; R² is acytotoxin; and L is a linker.
 2. The chimeric molecule of claim 1,wherein said cytotoxin is selected from a group consisting of PE4E andPE38QQR.
 3. The chimeric molecule of claim 1, wherein said linker is apeptide linker.
 4. The chimeric molecule of claim 1, wherein L or R², ifpresent, are attached to the carboxyl terminus of R¹.
 5. The chimericmolecule of claim 1, wherein L or R², if present, are attached to theamino terminus of R¹.
 6. A method of delivering an effector molecule toa cell bearing an interleukin 13 receptor (IL13R), said methodcomprising the step of contacting said cell with a chimeric moleculecomprising said effector molecule attached to a polypeptide comprisingthe amino acid sequence of SEQ ID NO:5.
 7. The method of claim 6,wherein said effector molecule is a cytotoxin selected from a groupconsisting of PE4E and PE38QQR.
 8. A method of inhibiting the growth ofa cell expressing an IL13 receptor (IL13R), said method comprisingcontacting said cell with a cytotoxic molecule comprising a cytotoxincovalently attached to a polypeptide comprising the amino acid sequenceof SEQ ID NO:5.
 9. The method of claim 8, wherein said cytotoxin isselected from the group consisting of PE38QQR and PE4E.
 10. The methodof claim 8, wherein said cell is a neoplastic cell.
 11. A pharmaceuticalcomposition comprising a pharmacologically acceptable excipient; and acytotoxic molecule comprising a cytotoxin covalently attached to apolypeptide comprising the amino acid sequence of SEQ ID NO:5.
 12. Thecomposition of claim 11, wherein said cytotoxin is selected from thegroup consisting of PE38QQR and PE4E.